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How to go about creating a stably-transfected cell line

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How to go about creating a stably-transfected cell line

Postby JustinNg » Mon Sep 17, 2012 9:26 am

Hi everyone, I am a PhD student and I'm new to these boards.

I'm using P19 mouse embryonal carcinoma cells in my research, and I differentiate them into neurons using retinoic acid. I wish to create a stable clone expressing a GFP-fusion neurotransmitter receptor, of either NMDA-R or AMPA-R, but I am not very clear about what should be done.

First off, how should I go about getting a suitable expression vector? I.e., what should I be looking for? I know that the vector should have a mammalian selection marker, and most mammalian expression vectors usually have a selection marker for bacterial systems too. What is the procedure usually done? Is it to grow up the vector in bacteria and then purify it and then transfect it into my cells of interest? Is there a more straightforward way e.g. obtaining/buying sufficient vector with the GFP-fusion insert and directly transfecting them into the P19 cells?

Any other suggestions on how I could go about getting a stable GFP-protein expressing P19 cells will be appreciated too!
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Postby JackBean » Sat Oct 06, 2012 12:34 pm

No experience with mammalian systems, but the bacterial selection is there because of the cloning. You will probably purchase empty vector and will need to clone into it your gene of interest. This will be done in bacteria to multiply and select vector with correct version of your gene.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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